In the United States, breast cancer is second in cancer-associated deaths among women. Similar to most cancers, it is the collaborative effects of multiple mutations and genetic alterations that lead to breast carcinogenesis. Thus, it has becoming increasingly apparent that the successful treatment of breast cancer will rely upon understanding how multiple pathogenic mutations and pathways interact. This in turn will provide the basis and rationale for future targeted therapies. The TP53 gene is the most frequently inactivated tumor suppressor gene in human breast cancers, with half of all TP53 mutations being attributed to dominant negative point mutations. TP53 encodes for p53, a protein that regulates many aspects of cell growth. In 2004, the Park lab identified PIK3CA, the gene encoding the catalytic domain of PI3K alpha, as the most mutated oncogene in human breast cancer. PI3Ks are lipid kinases that also regulate diverse cellular signaling pathways. The majority of PIK3CA mutations occur in three hotspots, which have been linked to carcinogenesis, making them ideal targets for therapeutic development. In this study, the PI proposes to create and characterize a panel of isogenic cell lines to model the progressive genetic events leading to breast carcinogenesis. She will use non-tumorigenic human breast epithelial cell lines and knock in TP53 and PIK3CA point mutations singly and in combination to precisely recapitulate pathogenic mutations that occur in human breast cancers. This in turn will lead to a deeper understanding of how breast cancers arise, and provide the underpinnings for future targeted therapies. Furthermore, the PI proposes that p53 modulates PI3K signaling by binding to the PIK3CA promoter and inhibiting PIK3CA transcription. The PI will investigate these hypotheses via the following aims: Specific Aim 1: Somatic cell gene targeting to determine the cooperative carcinogenic effects of cell lines harboring PIK3CA and/or TP53 mutations in vitro and in vivo. Specific Aim 2: Assessment of genomic instability in PIK3CA and/or TP53 mutant cell lines. Specific Aim 3: Determine if mutation of TP53 prevents p53-dependent inhibition of PI3K/AKT driven tumorigenesis in PIK3CA mutated cell lines The goal of the proposed research training program is to provide outstanding laboratory and translational/scientific training for the PI, Sarah Croessmann, a graduate student in the Pathobiology Program at Johns Hopkins. This will be achieved under the mentorship of Dr. Ben Ho Park and the Breast Cancer Research Program through a carefully constructed training program that evenly blends laboratory experience with formal and informal didactic learning. The proposed project will provide outstanding training for Ms. Croessmann and prepare her for a future career as a leader in biomedical research.